Evaluation of the Toxicity of Intravitreally Injected PLGA Microspheres and Rods in Monkeys and Rabbits: Effects of Depot Size on Inflammatory Response.

Purpose: Poly(lactic-co-glycolic) acid (PLGA) inserts have been successfully developed for the treatment of posterior eye disease as a means of reducing injection frequency of intravitreally administered therapeutics. PLGA microspheres are also of interest for the delivery of intravitreal drugs, since they offer the advantage of being easily injected without surgical procedures or large injectors. Methods: In the current study, the toxicity of PLGA microspheres and rods was investigated in nonhuman primates (NHPs) and rabbits. An in vitro assessment of cytokine responses to PLGA in peripheral blood mononuclear cells (PBMCs) and macrophages was also performed. Results: Intravitreal administration of 3, 10, or 12.5 mg/eye of PLGA microspheres in NHPs resulted in a severe immune response characterized by a foreign body response. Follow-up studies in the rabbit confirmed this finding for PLGA microspheres ranging in size from 20 to 100 µm. In contrast, administration of PLGA rod implants with a similar PLGA mass did not elicit a significant immune response. In vitro assays in PBMCs and macrophages confirmed proinflammatory cytokine release upon treatment with PLGA microspheres but not PLGA rods. Conclusions: These data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Formulation and delivery issues for monoclonal antibody therapeutics.

Antibodies can have exquisite specificity of target recognition and thus generate highly selective outcomes following their systemic administration. While antibodies can have high specificity, the doses required to treat patients, particularly for a chronic condition, are typically large. Fortunately, advances in production and purification capacities have allowed for the exceptionally large amounts of highly purified monoclonal antibodies to be produced. Additionally, genetic engineering of antibodies has provided a stable of antibody-like proteins that can be easier to prepare. Together, these advances have made antibody-based therapies one of the most commonly pursued pharmaceuticals in biotechnology pipelines. With this success, however, has come a series of technical challenges in the formulation of antibody-based materials to maintain sufficient stability in a variety of configurations and sometimes at particularly high concentrations. This review focuses on issues related to identifying and verifying stable antibody-based formulations.

A novel in vitro method to model the fate of subcutaneously administered biopharmaceuticals and associated formulation components.

Subcutaneous (SC) injection is becoming a more common route for the administration of biopharmaceuticals. Currently, there is no reliable in vitro method that can be used to anticipate the in vivo performance of a biopharmaceutical formulation intended for SC injection. Nor is there an animal model that can predict in vivo outcomes such as bioavailability in humans. We address this unmet need by the development of a novel in vitro system, termed Scissor (Subcutaneous Injection Site Simulator). The system models environmental changes that a biopharmaceutical could experience as it transitions from conditions of a drug product formulation to the homeostatic state of the hypodermis following SC injection. Scissor uses a dialysis-based injection chamber, which can incorporate various concentrations and combinations of acellular extracellular matrix (ECM) components that may affect the release of a biopharmaceutical from the SC injection site. This chamber is immersed in a container of a bicarbonate-based physiological buffer that mimics the SC injection site and the infinite sink of the body. Such an arrangement allows for real-time monitoring of the biopharmaceutical within the injection chamber, and can be used to characterize physicochemical changes of the drug and its interactions with ECM components. Movement of a biopharmaceutical from the injection chamber to the infinite sink compartment simulates the drug migration from the injection site and uptake by the blood and/or lymph capillaries. Here, we present an initial evaluation of the Scissor system using the ECM element hyaluronic acid and test formulations of insulin and four different monoclonal antibodies. Our findings suggest that Scissor can provide a tractable method to examine the potential fate of a biopharmaceutical formulation after its SC injection in humans and that this approach may provide a reliable and representative alternative to animal testing for the initial screening of SC formulations.

Bacterial toxins as tools for mucosal vaccination.

Several studies have demonstrated that the biological properties of secreted bacterial toxins could be harnessed for the induction of mucosal and systemic immunity following application at epithelial surfaces. Although the properties and potential application of several of these toxins will be discussed in this review, special focus will be placed on Pseudomonas aeruginosa exotoxin A (PE). A non-toxic form of PE (ntPE) into which antigenic epitopes can be integrated appears to be a particularly promising vaccination tool, which is able to cross the polarized epithelia of the gastrointestinal, respiratory and reproductive tracts and selectively target macrophages and dendritic cells.

Emerging technologies that overcome biological barriers for therapeutic protein delivery.

In the past decade, genomic research and the nascent field of proteomics have exponentially increased the number of potential protein therapeutic molecules for treating medical needs that were previously unmet. To realise the full clinical potential of many of the novel protein drug entities arising from these intense research efforts, emerging protein delivery technologies may be required. Advanced delivery technologies may offer the ability to overcome biochemical and anatomical barriers to protein drug transport, without incurring adverse events, to deliver the agent(s) at a certain desired rate and duration, to protect therapeutic macromolecules from in situ or systemic degradation, as well as increase their therapeutic index by targeting the drug action to a specific site. This review will cover a myriad of novel and emerging technologies that are directed at bypassing biological barriers and that have shown promise in advancing the therapeutic potential of protein drugs.

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration.

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

Sustained release formulations of rhVEGF165 produce a durable response in a murine model of peripheral angiogenesis.

Local delivery of therapeutic angiogenic agents that stimulate blood vessel formation represents a promising strategy for the treatment of peripheral vascular disease (PVD). At present, requirements for temporal and spatial parameters for localized delivery are unclear, with a variety of sustained delivery approaches being examined. Two polymer-based sustained formulations containing the 165 amino acid isoform of human recombinant vascular endothelial growth factor-A (rhVEGF(165)) were evaluated for their potential application in the treatment of PVD following intramuscular injection. Microspheres prepared from a 50:50 ratio of polylactic-co-glycolic acid (PLGA) and a gel of PLGA polymer solubilized in N-methyl pyrrolidone (PLGA:NMP) were each loaded with rhVEGF(165) and tested in vitro and in vivo. PLGA microspheres averaged ∼30 µm in diameter and contained 8.9% (w/w) rhVEGF(165), while the PLGA:NMP gel was formulated with varying amounts of spray freeze-dried rhVEGF(165) to result in final gel formulations having concentrations of 0.36, 0.72, or 3.6 mg/mL rhVEGF(165). In vitro release of rhVEGF(165) from PLGA microspheres showed ∼10% cumulative release by day 6, whereas the cumulative release of rhVEGF(165) from the PLGA:NMP gel matrices (0.65% w/w loading) was less than 0.25% at this same time point. While the in vitro release characteristics of these two sustained release formulations were broadly different, the plasma rhVEGF(165) concentration-time profiles following hind-limb intramuscular (IM) injection of these formulations in non-compromised rats revealed similar in vivo pharmacokinetics. Three-dimensional resin casts of vascular architecture were prepared at days 3, 7, 14, 21, 28, 60, and 75 following a single IM dosing of these sustained release microsphere and gel matrix formulations in the gastrocnemius muscle of immune-compromised mice. Scanning electron microscopic visualization of these vascular casts demonstrated spatial arrangement of capillary sprouts and vessel enlargement consistent with profound vascular changes occurring within 3 days of dosing that persisted for 2 months, approximately 1 month beyond the anticipated completion of rhVEGF(165) release from these sustained delivery formulations. Vascular re-modeling events were correlated with histological and immunohistochemical parameters attributed to known biological actions of rhVEGF(165) signaling. Together, these pharmacokinetic and pharmacodynamic results support the use of sustained release PLGA-based formulations for the local delivery of rhVEGF(165) to achieve a durable vascular re-modeling response.

Local tissue distribution and cellular fate of vascular endothelial growth factor (VEGF) following intramuscular injection.

Vascular endothelial growth factor (VEGF) is an extracellular matrix (ECM)-binding growth factor capable of driving neovascularization. VEGF can potentially be applied clinically via intramuscular (IM) injection to correct local ischemia associated with peripheral artery disease (PAD). As interactions with ECM elements and cognate receptors at the site of an IM injection define the local biology of VEGF and previous studies have only focused on systemic distribution measurements, we have established a method to monitor the local VEGF distribution and fate. Fluorescent-labeled VEGF was prepared that bound to ECM and activated a cognate receptor similarly to VEGF. Beginning by 2 h and becoming complete by 12 h following injection, fluorescence microscopy demonstrated the transition of labeled VEGF from an initial extensive interaction with ECM components to a focused labeling of vascular endothelial cells. Biochemical characterization verified the association of VEGF with ECM components and modification of endothelial cell function associated with vascular permeability changes known to accompany VEGF actions. Our data provide information concerning temporal and spatial VEGF fate and actions at the site of an IM injection that can help guide decisions regarding the identification of acceptable formulation strategies.